Earlier studies from our laboratory on randomly isolated transcriptional signals of mycobacteria had revealed that the -10 region of mycobacterial promoters and the corresponding binding domain in the major sigma factor are highly similar to their Escherichia coli counterparts. In contrast, the sequences in -35 regions of mycobacterial promoters and the corresponding binding domain in the major sigma factor are vastly different from their E. coli counterparts (M. D. Bashyam, D. Kaushal, S. K. Dasgupta, and A. K. Tyagi, J. Bacteriol. 178:4847-4853, 1996). We have now analyzed the role of the TGN motif present immediately upstream of the -10 region of mycobacterial promoters. Sequence analysis and site-specific mutagenesis of a Mycobacterium tuberculosis promoter and a Mycobacterium smegmatis promoter reveal that the TGN motif is an important determinant of transcriptional strength in mycobacteria. We show that mutation in the TGN motif can drastically reduce the transcriptional strength of a mycobacterial promoter. The influence of the TGN motif on transcriptional strength is also modulated by the sequences in the -35 region. Comparative assessment of these extended -10 promoters in mycobacteria and E. coli suggests that functioning of the TGN motif in promoters of these two species is similar.